This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A number of methods for the production of microspheres based on emulsion polymerization and aerosol synthesis are being developed at the University of New Mexico. These methods will be adapted for the formation of uniform populations of microbeads that are monodisperse in diameter and that incorporate known concentrations of luminescent lanthanides, such as terbium and europium complexes. Direct synthesis of mondisperse microparticles (as opposed to size fractionation of polydisperse particles) will be highly advantageous because of the relatively high cost of many lanthanide complexes and will provide sufficient throughput for the production of flow cytometry reagents. Because of the susceptibility of lanthanides reduction in luminescence yield by hydration, we will develop a method for their encapsulation in both polar inorganic and apolar organic microspherical hosts as described below. In most cases it may be advantageous to encapsulate lanthanide ions that are complexed to conjugated molecular "antennae" to increase photoluminescence cross-section [54]. A number of terbium and europium complexes are commercially available (Aldrich, Strem) as are lanthanide labels. (Invitrogen) that have a range of excitation characteristics. Encapsulation of Lanthanides in Inorganic Hosts. Our first aim will be to develop methods for the facile and reproducible production of monodisperse silica particles that encapsulate luminescent lanthanide ions in well-defined concentrations. Lopez, et al. at UNM have recently developed sol-gel methods for production of monodisperse silica microparticles from aerosol droplets (see Fig. 14). [55] In this method, which has been shown to be conducive to the incorporation of inorganic and organic hosts in the particles, uniform aerosol droplets with the desired precursors are generated using a vibrating orifice aerosol generator. Using this method we will introduce known concentrations of photoluminescent terbium and europium complexes. We will optimize the molecular level dispersal and total particle lumimescence to enable direct detection of lanthanide photoluminescence using new acoustically focused flow cytometers developed by the NFCR. These silica-based beads will be readily amenable to surface biofunctionalization by established silane based coupling chemistry [56]. Encapsulation of Lanthanides in Organic Polymeric Microbeads. Because of the issue identified above related to the potential low luminescence of hydrated lanthanides, our second aim will be to develop methods based on emulsion polymerization of organic monomers for the facile preparation of monodisperse particles that incorporate lanthanide ions. Emulsion based polymerizations are well known, but bulk emulsion methods generally result in particles with polydisperse sizes (generally in a log-normal size distribution) [57]. For flow cytometry applications it is highly desirable to use particle populations of uniform size, and thus, we will develop methods for direct formation of monodispersed polymeric particles encapsulating lanthanides. To do so we will use microfluidic injection methods for forming stable monodisperse emulsion droplets developed at Harvard University [58]. Through an NSF funded collaboration, the methods for forming such monodisperse droplets have recently been transferred to UNM. The droplets shown in Fig. 14 were generated in microfluidic devices fabricated and utilized at UNM. We will develop methods of stabilization of such emulsion droplets, of loading them with organic monomers such as styrene, methyl methacrylate and n-butyl methacrylate, together with luminescent lanthanide complexes, and initiating polymerization reactions to form uniform microspheres. Such emulsion polymerization methods for forming microspheres are especially powerful because of the ability to form core-shell architectures in which are surface coated with biomolecule-reactive functional groups in one step.